White-rot fungus and uses thereof

ABSTRACT

The white rot fungus Scytinostroma galactinum strain F361 and mutants thereof are particularly effective in selectively grading the lignin component of lignin-containing materials, particularly processed wood pulps including chemical pulps, and also particularly effective in degrading lignin degradation products such as chlorinated degraded lignin by-products as found, for example, in E-1 effluents, and also in degrading chlorine-containing aromatic compounds generally as found in aqueous waste streams containing the same.

This is a continuation of application Ser. No. 08/198,459, filed Feb.18, 1994, now U.S. Pat. No. 5,427,945 which in turn is a continuation ofapplication Ser. No. 07/276,081, filed Nov. 23, 1988, now abandoned.

This invention relates to fungi of the white rot type and the usethereof, particularly in the modification and degradation of lignin andmodified lignins.

Various bacteria and fungi are known to survive on and degrade wood. Forexample, white-rot basidiomyctes (fungi) are known to form and surviveon wood, and their nourishment as obtained from the wood is manifestedin the observed slow decay of the wood. Such fungi typically obtaintheir nourishment from the ultimate degradation products of cellulosebut release a host of enzymes which degrade substantially all organicwood components including not only the cellulose but also hemicelluloseand lignin.

White-rot basidiomycetes remove lignin from wood in severalmorphologically distinct patterns. One type of decay known as "selectivedelignification" is apparent when greater amounts of lignin are degradedrelative to the amount of cellulose. In this type of decay, lignin inthe secondary wall and middle lamella may be almost entirely removed,whereas large quantities of cellulose in the S2 layer of the cell wallare left intact. White rot basidiomycetes can also cause a "simultaneousrot". This type of decay is characterized by the removal of bothcellulose and lignin, leaving cells either fiddled with bore holes anderosion troughs, or with extensively thinned secondary walls. Muchvariation exists among these decay types. Some Basidiomycetes cause onlya simultaneous rot whereas others may produce a simultaneous rot in onepart of the substrate and predominantly a delignification in another. Insuch cases, a chemical analysis of the entire substrate can misrepresentthe potential of these fungi to cause selective lignin removal. Someother white rot fungi have been shown to be initially very selective forlignin and then later attack the remaining cellulose. Thus, theselectivity of some fungi for lignin can change depending on the stageof decay at which chemical analyses are done.

To avoid difficulties arising from the interpretation of lignin and woodsugar analyses, several methods are used to evaluate the capacity offungi to selectively degrade lignin. A screening technique involvingscanning electron microscopy has been used to differentiate the type ofdecay produced by different white rot fungi. This technique, coupledwith traditional chemical analyses, provides the more accurate means ofscreening available.

One such type of white rot fungi, Phanerochaete chrysosporium andmutants thereof (see, for example, U.S. Pat. No. 4,687,741) can beviewed with simplicity as releasing a battery of largely cellulosedegrading enzymes (cellulases) in order to obtain its nourishment fromwood. Since the cellulose is isolated within barrier layers or regionsmade up of hemicellulose and lignin, the supply of cellulose directlyavailable to the fungus is limited and becomes exhausted. At this pointthe fungus has less and less cellulose available for survival and entersessentially a starvation phase in which the fungus then releaseshemicellulose and lignin degrading enzymes (hemicellulases andligninases, respectively) which remove the barrier layers and expose thenext layered supply of cellulose for nourishment. This cycle will berepeated on and on in the process of the total natural decay of wood.Essentially on the theory that the fungus could be induced to produceenzymes affecting lignin in deference to those affecting cellulose,Phanerochaete chrysosporium cultures were found to degrade lignins inpulps, including chemically modified pulps (known as chemical pulps), ina selective manner. The fungus was also found to degrade chemicallyaltered lignin degradation products as produced, for example, in theprimary bleaching stage of the kraft process (so-called "E-1effluents"). P. chrysosporium is more effective on hardwood lignins thanon softwoods.

One primary chemical method for making pulp from wood involves thedigestion of lignin in the wood with sodium sulfide and sodiumhydroxide. This is termed the sulfate or kraft process, and theresulting pulp is termed "chemical pulp". The known "sulfite" processalso produces a chemical pulp.

Wood pulp produced, for example, in the kraft process, generallycontains about 4-12% by weight of residual lignin, at least some ofwhich is chemically modified to give the pulp a characteristic browncolor. To obtain a pulp of high brightness and brightness stability, thelignin and/or modified lignin must be further treated and removed bycertain agents in one or more treatments or stages commonly referred toas bleaching. And, in general, all chemical pulps are subjected to oneor more bleaching treatments for similar purposes. Many bleachingprocesses exist but almost all kraft pulp bleachings begin with thechlorination-extraction (C-E) stage. Other bleachings use hypochlorite,chlorine dioxide, oxygen or other oxidizing chemicals such that thedegraded lignin component freed from the pulp is chlorinated and/oroxidized, and hence further chemically modified. There is some loss anddamage of cellulosic fibers during the C-E stage but the cellulosiccontent remains high and sufficiently intact that paper/board productsof acceptable quality can be produced, hence the process is selective.The C-E effluents (included among those called E-1 effluents) resultingfrom treated pulp contain a very large number of organic compoundshaving a bound chlorine content of 2.5-3.5 kg/ton pulp. Some of thesecompounds, primarily the chlorinated phenolics, have been identified ashaving toxic, mutagenic and carcinogenic effects (Alberti, B. N. andKlibanov, A. M. [1981] Biotechnology and Bioengineering Symp.11:373-379). Alternatives to chlorine bleaching have, therefore, longbeen sought by industry. As use herein, the term E-1 effluent refers tothe effluent resulting from extraction of chemical pulp after the firstor primary bleaching stage.

The use of fungi and their enzyme systems in the processing of woodpulps and chips (biopulping generally) to improve efficiency and reduceenvironmental problems in the pulp and paper industry has been thesubject of increasing interest and investigation.

The major object of the present invention is to provide a fungus withparticularly suitable properties and capabilities for degrading ligninin a manner which is selective in lignin relative to affecting thecellulose content of the substrate. Another major objective is toprovide a fungal system capable of degrading chemically modified ligninssuch as those presented in chemical pulps, and particularly thosepresented in the kraft pulping process immediately prior to or at a timeafter bleaching. A further object is to provide a fungus for degradingchemically modified lignin degradation products such as those ineffluents (eg. the so-called E-1 effluent) from the pulp bleachingprocesses.

These and other objects will be apparent from the following description.

In accordance with the present invention it has been found that a strainof Scytinostroma galactinum, identified as strain F361, is particularlyeffective in selectively degrading lignin and chemically modifiedlignins, and also in degrading lignin degradation products, includingchemically modified lignin degradation products. Such ability of F361 isremarkable since this fungus appears to produce only small relativeamounts of the type of ligninase enzymes which made systems based onPhanerochaete chrysosporium of interest for similar uses. The fungusF361 has also been observed to be relatively slow growing in liquidculture but is surprisingly effective in selectively degrading lignin inwood. It is particularly desirable in that it selectively andsignificantly degrades lignin in both softwoods and hardwoods. TheFungus F361 is also capable of growing for extended periods and its longlife is of particular advantage, especially in the degradation ofchemically modified lignin degradation products found in waste streamssuch as the E1 effluent from the kraft process.

The Scytinostroma galactinum strain F361 was deposited under theBudapest Treaty as a biologically pure culture on Nov. 2, 1989 with theATCC and received the accession No. ATCC 20966. Since this strain maynaturally undergo mutation or mutation may be artificially inducedwithout changing the desirable or advantageous characteristics of F361,the invention is also deemed to apply mutants of F361 which retain thebasic properties and capabilities of the F361 strain.

The term "lignin component" as used herein is designed to refer to notonly natural lignins which have not been chemically modified but alsothe form in treated or processed pulps wherein the lignin is chemicallymodified by various chemical treatments such as in the pre-bleachingstage of the Kraft process.

The term "degrading" as used herein with reference to the lignincomponent in a substrate is meant to encompass not only fungal enzymeaction which modifies or degrades the lignin component in a mannercausing it to dislodge either immediately or upon washing or extractionof the substrate but also fungal enzyme action which modifies ordegrades the hemicellulose which binds or holds the lignin component,which action results in or enables the freeing of the lignin componenteither immediately or upon washing or extraction of the treatedsubstrate, eg. pulp. Hence, the degradation of lignin as encompassed bythe invention is an action essentially effective to release the lignincomponent from the substrate. In the case of many pulps, the desiredaction is evidenced by a brightening of the pulp.

The F361 fungal strain and mutants thereof may be grown in a knownmanner using, for example, stationary flasks or a Rotating BiologicalContactor (RBC). Since the fungus is slow growing, an extended timeperiod is generally allowed. Growth of the F361 fungus is described indetail in Example 1, hereinafter. The fungus as a biologically pureculture is generally grown in the presence of conventional fungal growthmedia including a carbon source such as glucose. To degrade a lignincomponent and its by-products, the fungus is desirably cultured in thepresence of an inducer in order to encourage or enhance production bythe fungus of its lignolytic enzymes which effect the desireddegradation of the lignin component or lignin degradation products whichthe fungus degrades. Suitable inducers are lignin, lignin-containingsubstances and substances having an origin in lignin including itsdegradation products, both natural and those arising from chemicaldegradations. Examples of such inducers include isolated and purifiedlignin, cellulosic substrates containing a bound lignin component andaromatic ring-containing lignin component degradation products which areunbound to a cellulosic substrate. Suitable inducers therefore includeunprocessed wood, in various forms such as block, chips or dust,processed wood such as pulps in which the lignin is untreatedchemically, various modified pulps including kraft or other chemicallytreated pulps, lignin itself, E1 effluent or other waste streamscontaining chemically modified lignin degradation products and so-calledaromatic lignin model compounds which represent natural degradationproducts of lignin which are further modified or degraded by the fungus,such as 2,6-dimethoxyphenol or other hydrocarbon aromatic ringsubstituted by a hydroxy group, particularly a phenol, or veratrylalcohol. The chemical compounds which the fungus also modifies ordegrades but which do not have an origin in lignin may also be used asinducers although it is generally preferred to induce the desired growthphases with substances having an origin in lignin. It will beappreciated that different inducers may induce at any given growth timevarying proportions of the lignin degrading enzymes produced naturallyby the fungus. In general, inducers are used in cultural growth in thepresence of suitable growth media at approximately a 1% level weight byvolume. On the other hand, the inducer may be present in highproportions to effect a desired action on the inducer by way of achemical modification or degradation. An inducer for hemicellulases mayalso be added, particularly an inducer for mannanases when softwood isbeing treated. Substances having an origin only in cellulose, such asglucose, while useful in growing the fungus, are indicated to have asignificant inhibitory effect on production by the fungus of desiredenzymes and therefore are desirably used in reduced amounts oreliminated entirely when culturing the fungus for practical applicationssuch as in the bleaching of pulp.

In general, substances which induce the production of lignin degradingenzymes by the fungus are also substances which may be desirablymodified or degraded by the fungus.

The F361 strain of S. galactinum may be used in accord with theinvention in any of a variety of ways to modify or degrade lignin andlignin degradation products, including both natural lignins and theirdegradation products and modified lignins; particularly chemicallymodified lignins, and their degradation products. A suitable typesubstrate is a cellulosic substrate containing a bound lignin component.Typically, as is the case with wood, the binding is associated withhemicellulose which is also contained by such substrates. The fungus isparticularly useful in a variety of applications on wood pulp. Variousmechanical pulps are of interest (see Casey, J. P. (1983) Tappi Journal66:95-96). Hence, the fungus may be used to treat thermomechanical pulps(TMP) to reduce its lignin component to enhance brightness stability andenhance strength properties. Other mechanical pulps such aschemithermomechanical pulp (CTMP) and chemimechanical pulp (CMP) may bealso treated with the fungus to improve brightness stability andstrength properties. Of particular interest for treatment by the fungusare chemical pulps such as sulfite pulps and kraft pulps which may bebleached by the fungus to reduce the lignin component content,particularly in the stage of the kraft process in which chlorinebleaching has been used.

Another use of particular interest involves the treatment of aqueouseffluents or waste streams comprising a degraded lignin component inorder to further degrade the lignin component. More particularly ofinterest for such treatment are the effluents discharged from pulpingprocesses and containing chemically modified, degraded lignincomponents, especially those from the kraft process such as the E1effluent containing aromatic substrates resulting from the chlorinebleach stage. The treatment of such effluents by the fungus degrades thechloro-aromatic lignin degradation products and effects a lightening ordecolorizing of these normally brown or discolored liquids.

In general, cellulose/hemicellulose/lignin substrates may be treatedwith F361 or mutant thereof to selectively reduce the lignin content byinoculating the substrate or media containing the substrate with thefungus such that the fungus grows in contact with the substrate.Temperatures may range from 15° C. to 40° C., and are preferably in therange of 20° C. to 34° C. Oxygen or an oxygen-containing gas such as airis generally provided to support fungal growth. In the treatment of pulpsubstrates such as kraft pulps with the fungus to effect a substantialremoval of the lignin component and a bleaching thereof, it has beenindependently found particularly effective to employ the fungus insystems in which the pulp consistency is in the medium to high range,hence a pulp consistency of at least 7%, more preferably at least 9% anddesirably at least 12%. Pulp consistencies may range as high as 30% oreven somewhat higher but are more suitably in the range of 10% to 20%.Consistency is determined by the standard procedure involving the dryweight of pulp (after drying for 2.5 hours at 60° C.) as a percentage ofthe total system measured in volume.

The time of treatment sufficient to modify or degrade the lignin mayvary in the substrate widely from a few hours to several days dependingupon a number of factors such as the extent of treatment desired, thesubstrate being treated and the conditions and efficiency of the systememployed. The pH of such systems is suitably not greater than 7, andusually in the range from 3 to 6, preferably 4 to 5.

For the treatment of substrates normally encountered in diluted media,such as degraded lignin-component containing waste streams or effluents,eg. E1 effluent, these are usually treated by contacting the effluentsolution or suspension with the fungus as grown on slants, plates, discsand the like, such as demonstrated herein or as described in U.S. Pat.No. 4,554,075. Temperatures are generally in the range of from 15° C. to40° C., preferably 20° C. to 34° C. The reaction system is continuouslyor periodically supplied with oxygen in a manner such that the oxygen isavailable to the fungus. Pure oxygen or an oxygen containing gas such asair may be used. Preferably, the contact of the waste stream with anygiven portion of the fungal culture is intermittent such that theculture is exposed alternately to the effluent and oxygen. Analysis ofthe degraded E-1 effluent from the chlorine bleaching of kraft pulpindicates an ability of the fungus to essentially remove chlorine fromthe aromatic components thereof, as revealed by an increase in inorganicchlorides and reduction in organic chlorides. It is therefore furtherindicated that the F361 fungus may also be used to degrade otherunwanted chlorine-containing, aromatic compounds (eg. ring substitutedby chlorine) generally as may be found in aqueous waste streamscontaining the same. Such a treatment, which may be carried in the samemanner as E-1 effluents are treated, are also within the scope of theinvention.

The following examples are merely illustrative of the invention andfactors bearing on its practice.

EXAMPLE 1 Growth of Scytinostroma galactinum

a) Preparation of mycelial suspension

The F361 mycelia are grown on Yeast Nitrogen Phosphate Dextrose (YNPD)agar with the following nutrients:

    ______________________________________                                               glucose        10 gms                                                         malt extract   10 gms                                                         peptone         2 gms                                                         yeast extract   2 gms                                                         asparagine      1 gm                                                          KH.sub.2 PO.sub.4                                                                             2 gms                                                         MnSO.sub.4.7H.sub.2 O                                                                         1 gm                                                          thiamin.HCl     1 mg                                                   ______________________________________                                    

in a final volume of one liter; the pH adjusted to pH 4.2. The mycelialfragments are inoculated onto slants containing 50 ml YNPD agar whichare loosely capped and remain at room temperature. Growth occurs in 3 to4 weeks. A mycelial suspension is prepared by diluting the cultures with100 ml of sterilized deionized water, mixing well and filtering throughsterilized cheesecloth.

b) RBC Growth in Presence of Veratryl Alcohol

Two liters of the fungal growth media in the RBC is inoculated with themycelial suspension from the slant tube, and left to grow at roomtemperature, 21° to 25° C. The RBCs are rotated at one revolution perminute. Establishment of fungal cultures onto the discs takes one month.The fungal cultures are aerated 3 to 4 times per week with oxygen, byflooding the airspace over the culture with pure oxygen. Media isreplenished as it evaporates, with 1 to 2 changes of media during thefirst 2 months.

The growth media consists of the following nutrients, buffers andelements:

2 ml of 0.4M veratryl alcohol

200 ml of 10×B-3

200 ml of 10% glucose

200 ml of 0.1M trans-aconitic acid (recrystallized)

120 ml of 100×trace elements

200 ml of the mycelia suspension

100 ml of H₂ O

The 10×B-3 is composed of:

    ______________________________________                                        NH.sub.4 tartrate        2 gms                                                KH.sub.2 PO.sub.4       20 gms                                                MgSO.sub.4.7H.sub.2 O    5 gms                                                CaCl.sub.2               1 gm                                                 thiamin.HCl             10 mgs                                                100 × trace elements                                                                            70 mLs                                                ______________________________________                                    

and deionized H₂ O to equal 1000 mLs total volume. The 100× traceelement solution contains the following:

    ______________________________________                                        nitriloacetate       1.5    gms                                               MgSO.sub.4.7H.sub.2 O                                                                              30     gms                                               MnSO.sub.4           0.5    gms                                               NaCl                 10     gms                                               FeSO.sub.4.7H.sub.2 O                                                                              0.1    gm                                                COSO.sub.4           0.1    gm                                                CaCl.sub.2.2H.sub.2 O                                                                              0.1    gm                                                ZnSO.sub.4           0.1    gm                                                CuSO.sub.4.5H.sub.2 O                                                                              0.01   gm                                                AIKSO.sub.4          0.01   gm                                                NaMoO.sub.4          0.01   gm                                                Boric acid           0.01   gm                                                ______________________________________                                    

in one liter total volume of deionized water. Boric acid is dissolved in200 ml of deionized water, the pH adjusted to 6.5 with KOH, and thevolume is brought to 600 ml. Each mineral is added in the above orderand dissolved before the next addition.

EXAMPLE 1-A

Stationary flasks of F361 are grown under the same conditions as Example1 with approximately 50 ml of growth media in a 250 ml flask or 250 mlof media in a 2 liter flask.

It was also observed that cultures grown at room temperature in astationary flask on minimal media (containing 192 μM of solublemanganese salt) in the presence of veratryl alcohol produced after about25 days clearly recognizable quantities of a black material which wasidentified as manganese oxide. This is the first known instance of afungal liquid culture producing manganese oxide in the absence of wood.Analysis of enzymes from the supernatant of cultures of F361 has shownrelatively high proportions of manganese dependent peroxidases, aheme-containing oxidative enzyme which has been associated with lignindegradation. Similar analysis has also shown the presence of laccase.

EXAMPLE 2

The experiment of Example 1 is repeated except that, after about 10weeks of growth, 2 liters of growth media containing trace elements andan E-1 hardwood effluent obtained from the S. D. Warren Company SomersetMill was added to the RBC. The E-1 effluent was diluted with the 2liters of growth media as described in Example 1, part b), to anabsorbance 0.436 (465 nm) which appeared as the color of weak tea. ThepH was adjusted to 4.5 and with the E-1 effluent growth media sterilizedat 121° C. prior to being added to the RBC. After two days the color wasreduced to an absorbance of 0.133 absorbance units (growth media wasused to blank the spectrophotometer at 465 nm), a reduction of 70%color, indicating a substantial decolorization of the E1 effluent by thefungus.

EXAMPLE 3 Decolorization of Hardwood E-1 Effluent with Time

To monitor the hourly decrease in color of E-1 effluent the followingexperiment was carried out. A 1:3 dilution of E-1 effluent (one parteffluent to three parts growth media) is adjusted to pH 4.5 with HCl andautoclaved at 121° C. for 30 minutes and cooled to room temperature. Twoliters of this 1:3 dilution is added to the same RBC used in Example 2.The absorbance of an aliquot removed hourly for the first 10 hours wasread at 465 nm versus the blank which was growth media alone. Theresults are given in the following table A:

                  TABLE A                                                         ______________________________________                                               Hour(s)                                                                              A465 nm                                                         ______________________________________                                               0      0.717                                                                  1      0.595                                                                  2      0.535                                                                  3      0.485                                                                  4      0.464                                                                  5      0.445                                                                  6      0.421                                                                  7      0.399                                                                  8      0.381                                                                  9      0.375                                                                  14     0.320                                                                  30     0.255                                                                  73     0.175                                                           ______________________________________                                    

During this time the fungal mat on the RBC disc was becoming mottled inappearance with light and dark areas. Normally, the mat is a creamywhite color. Eventually, the mat turned a dark brownish color. Todetermine if colored products were being absorbed by the mycelia or if amelanin pigment was being produced, sections of the mycelia wereanalyzed. The dark brown areas were deposits of manganese oxidecrystals. Elemental analysis of the crude, undiluted E-1 effluent andeffluent diluted with growth media showed that the manganese had comefrom the growth media and not from the E-1 effluent. Crude E-1 effluentcontains only 0.63 ppm manganese whereas the growth media contains 13ppm.

EXAMPLE 4 Hardwood E-1 Effluent Saturation Study

Hardwood E-1 effluent obtained from the Somerset Mill, Me., USA, wasdiluted 1:2 (growth media: E-1 effluent) with fungal growth media, thepH adjusted to 4.5 with HCl, and the solution autoclaved at 121° C. for30 minutes. After cooling, 2 liters of the E-1 effluent-media mixturewas added to a RBC containing F361 which had been exposed to hardwoodE-1 effluent for 25 days. (After the experiment in Example 3 wascompleted, the growth media effluent mixture was left in the RBC untilremoved for this experiment). The RBC rotated at one revolution perminute on the bench top at room temperature. The air-space over thesolution was flooded with 100% oxygen for 10 minutes, 3 times a week.The sterilized growth media-effluent solution was changed daily withfresh solution prepared as described above. The decrease in color wasmonitored at 465 nm after exposure for 24 hours to the fungal mat.Growth media alone is used to blank the spectrophotometer. The resultsare given in the following Table B:

                  TABLE B                                                         ______________________________________                                        day       A465 nm  % Absorbance decrease                                      ______________________________________                                        0         1.460    0                                                          1         .780     47                                                         2         .828     40                                                         3         .922     37                                                         4         .899     37                                                         5         .948     38                                                         6         .937     35                                                         7         .922     37                                                         ______________________________________                                    

EXAMPLE 5 Softwood E-1 Effluent Decolorization

Softwood E-1 effluent obtained directly from the bleach plant at S. D.Warren Co., Somerset Mill, Me., USA, was diluted 3:1 (E-1 effluent tofungal growth media), the pH adjusted to 4.5 with HCl and autoclaved for30 minutes at 121° C. The RBC used in Example 4, which is now 4 monthsold, two of those months it has been constantly exposed to hardwood E-1effluent is used in this experiment. After cooling to room temperaturetwo liters of the softwood E-1 effluent are added to the RBC and the RBCis continued to be aerated with 100% O₂ for 10 minutes, three times aweek. Decolorization of the E-1 effluent is followedspectrophotometrically as described in Example 3. The results are givenin the following Table C:

                  TABLE C                                                         ______________________________________                                        time (days) A465 nm  % Absorbance decrease                                    ______________________________________                                        0           2.486                                                             1           1.266    49                                                       11          0.848    66                                                       ______________________________________                                    

EXAMPLE 6 Softwood E-1 Effluent Saturation Study

The saturation study was carried out with the RBC used in Example 5, andthe same dilution of softwood E-1 effluent as described there. The E-1effluent growth media mixture was replaced daily with fresh 1:3 mixture.The absorbance decrease at 465 nm was recorded after 24 hour exposuresto the fungal mat. The results are shown in the Table D:

                  TABLE D                                                         ______________________________________                                        day       A465 nm  % Absorbance decrease                                      ______________________________________                                        0         2.788    0                                                          1         1.9957   28                                                         2         2.3538   16                                                         3         2.3458   16                                                         4         2.1223   24                                                         5         2.2769   18                                                         6         1.9461   30                                                         7         2.0083   28                                                         ______________________________________                                    

EXAMPLE 7 Treatment of Undiluted Softwood E-1 Effluent

A comparison between a new growth of F361, 1 month old, and the F361 RBCused in Example 7, now 5 months old (3 months exposed to E-1 effluent)was made to see which growth would decolorize undiluted softwood E-1effluent at pH 4.5 and pH 10.5. Both growths were exposed to a 3:1dilution of E-1 effluent to growth media as prepared in Example 5 for 2days. The 3:1 dilution was removed and replaced with 2 liters undilutedE-1 effluent, pH 4.5 which was previously adjusted to pH 4.5 with HCl,autoclaved for 30 minutes at 121° C., and cooled. Results are given inTable E:

                  TABLE E                                                         ______________________________________                                               1 month old RBC                                                                             5 month old RBC                                          (hours)  A465    % absorb. dec.                                                                            A465  % absorb. dec.                             ______________________________________                                        3:1 dilution                                                                   0       3.3053              3.3053                                           24       3.1704  4           3.0316                                                                              8.3                                        48       3.1503  5           2.9333                                                                              11.2                                       undiluted                                                                     E-1 effluent                                                                   0       3.3086              3.3086                                           24       3.3426              3.2876                                                                              0.6                                        48       3.3372              3.2631                                                                              1.4                                        ______________________________________                                    

The undiluted, pH 4.5, softwood effluent was replaced in each RBC withtwo liters E-1 effluent as it comes straight from the paper mill,undiluted, pH not adjusted and not autoclaved. The decrease in color wasmonitored over the next 8 days. No decrease in absorbance was observedfor either RBC, and during the first 24 hours the pH of the E-1 effluentdropped from 10.3 to 9.2 with the new growth of F361 and from 10.3 to8.6 with the old growth. The fungus started coming off the RBC discsafter 8 days.

EXAMPLE 8 Results of Action of F361 Grown with Glucose on Kraft Pulp

a) The action of F361 on the kraft pulp added to the RBC of Example 1was monitored by periodic visual observation over the course of fourweeks time after addition of the pulp with no significant bleachingeffect on the pulp.

b) Stationary flasks of F361 are grown under the same conditions asExample 1-A with approximately 150 ml of growth media in a 250 ml flaskto which was added 1.5 grams dry weight of Southern softwood kraft pulpor Northern hardwood kraft pulp (pulp consistency about 1.0%). Theflasks were autoclaved at 121° C. for 30 minutes and cooled prior toinoculation with 30 ml of a mycelial suspension prepared as inExample 1. The flasked are cultured at room temperature and aerated withpure oxygen 4 times a week. After 30 days time at 25° C., no significantbleaching was observed and Kappa measurements showed no reductionsoutside of experimental error. The failure to attain a bleaching effectin the experiments of this Example 8 was attributed mainly to aninhibitory effect by glucose on the timely production of desiredenzymes.

EXAMPLE 9 Bleaching of Kraft Pulp with F361

A series of stationary flask (250 ml) experiments were set up with F361inoculated directly onto hardwood or softwood kraft pulps obtained fromthe Scott Paper Company. The pulps were water washed, filter dried andsamples divided in two sets. To one set 20 ml of growth media (seeExample 1) was added to the pulp before inoculation. To the other setonly 20 ml of water was added to the pulp before inoculation. Afterautoclaving inoculation were effected by adding to the flasks equal sizecubes of agar plugs obtained from YNPD agar slants of F361. The hardwoodkraft pulp experiments were conducted at a pulp consistency of 23% (dryweight pulp to vol.) and the softwood experiments were conducted at apulp consistency of 9% (dry weight pulp to vol.). The dry weight of thepulp was determined by the standard procedure of drying for 2.5 hours at60° C. By day 21 a very heavy bleaching had been observed in all samplesand on day 26 (after inoculation) the samples were recovered and treatedby a standard alkali extraction (2.5% aqueous sodium hydroxide at 60°C). Kappa numbers were then determined relative to controls and theresults reported below in Table F.

                  TABLE F                                                         ______________________________________                                        Treated Kraft Pulp                                                                           Kappa   % Kappa Reduction                                      ______________________________________                                        Softwood                                                                      Grown in media 6.2     64                                                     Grown in water 5.6     67.6                                                   Control        17.3    --                                                     Hardwood                                                                      Grown in media 6.1     52                                                     Grown in water 5.5     56.7                                                   Control        12.7    --                                                     ______________________________________                                    

EXAMPLE 9-A

The experiment Example 9 was repeated except that fungal samples grewfor 32 days, after which kappa was measured. For this experiment, sincethe fungal grown samples were visually so well bleached, samples weredivided into two parts. To one set the samples were alkali extracted, soas to solubilize lignin, as is normally done by the pulp and paperindustry. The other set the samples were washed in hot water but notalkali extracted. The results are given below in Table G.

                  TABLE G                                                         ______________________________________                                        Treated Kraft Pulp     Kappa                                                  ______________________________________                                        Softwood                                                                      Softwood control & alkali extracted                                                                  17.3                                                   Grown in media & alkali extracted                                                                    4.5                                                    Grown in media & water washed                                                                        9.4                                                    Grown in water & alkali extracted                                                                    5.8                                                    Grown in water & water washed                                                                        6.4                                                    Hardwood                                                                      Hardwood control & alkali extracted                                                                  12.7                                                   Grown in media & alkali extracted                                                                    6.5                                                    Grown in media & water washed                                                                        9.3                                                    Grown in water & alkali extracted                                                                    5.4                                                    Grown in water & water washed                                                                        5.6                                                    ______________________________________                                    

EXAMPLE 10

Northern softwood (pine) kraft pulp and Northern hardwood kraft pulp,both obtained from the Warren Somerset Mill (Me., U.S.A.) were eachindividually evaluated in a series of bleaching experiments using theF361 strain of Scytinostroma galactinum. The pulp was cleaned by washingwith deionized water and filtered to give a pulp of about 30%consistency. Samples were dried for 2.5 hours at 60° C. to determine thedry weight of the pulp in the moist pulp. Five (5) grams by dry weightof the moist pulp was placed in each of several 8 inch by 10 inchautoclave polypropylene bags and varying amounts of water added to thebags to provide a duplicate series of bagged samples having a differentpulp consistency. The bags were sealed and autoclaved at 121° C. for 35minutes to sterilize the pulp samples, and each then cooled to roomtemperature. Each bag in a duplicate series was then inoculated withabout an equal amount of an agar cube of F361 and the bags agitated todistribute the fungal inoculum in the pulp (the other duplicate seriesbeing maintained as a control to which no inoculum was added). Asterilized 5 inch long pasteur pipet fitted with a small sterilizedcotton plug was then inserted into each bag and the bag opening sealedaround the pipet. Each bag was then aerated through the pipet with pureoxygen and the bags allowed to rest on the work bench top at roomtemperature. Each day during the experiment each bag was recharged withoxygen. After 19 days, the pulp was recovered from the bags and both thetreated samples and untreated control samples were extracted with sodiumhydroxide in the conventional manner and the Kappa number of each sampledetermined, and the results averaged for each pair of samples at eachconsistency. The results are reported below in Table H.

                  TABLE H                                                         ______________________________________                                                       Kappa No.                                                      Pulp Consistency (%)                                                                           F361 Treated                                                                             Control                                           ______________________________________                                        Softwood    1        19.9       21.0                                                      3        16.8       19.7                                                      6        17.8       19.2                                                      9        13.7       18.9                                                      12       12.9       19.3                                                      15       7.3        20.5                                                      20       12.4       19.8                                          Hardwood    1        12.0       13.6                                                      3        12.5       13.1                                                      6        13.1       12.6                                                      9        10.5       12.7                                                      12       6.7        13.0                                                      15       6.9        13.9                                                      20       7.1        14.1                                          ______________________________________                                    

EXAMPLE 11

Example 10 was repeated using the same pulps obtained from the samesource but at a different time. The results are reported below in TableI.

                  TABLE I                                                         ______________________________________                                                       Kappa No.                                                      Pulp Consistency (%)                                                                           F361 Treated                                                                             Control                                           ______________________________________                                        Softwood    9        4.3        15.7                                                      12       4.9        15.7                                                      15       5.6        15.7                                                      18       3.7        15.7                                                      21       4.6        15.7                                          Hardwood    3        12.4       20.8                                                      5        15.6       19.1                                                      6        12.9       18.2                                                      7        10.2       21.4                                                      9        7.9        19.8                                          ______________________________________                                    

EXAMPLE 12

Stationary flask experiments in the general manner of Example 8(b) werecarried at 4.5% and 16.5% pulp consistencies with xylan and/or mannan asan inducer for xylanase and mannase enzymes to remove hemicellulose fromkraft pulp. A preinoculum for the hardwood pulp was prepared by mixingfor 4 days on a shaker table 10 ml of an aqueous suspension of F361mycelial fragments (obtained from a stationary flask) together with apreinoculum media composed of 0.25% xylan, 1.0% E-1 effluent fromhardwood pulp bleaching and 90 ml of a modified 1× concentrate of theF361 growth media prepared as in Example 1 except that the veratylalcohol was omitted and only 0.4% of glucose was added (total 100 ml in500 ml flasks). A similar preinoculum was prepared for softwood exceptthat 0.5% mannan was substituted for the xylan and a softwood E-1effluent was substituted for the hardwood effluent. All pulps weresterilized prior to inoculation.

Hardwood kraft pulp was treated in one series of 500 ml flasks by mixing5 grams dry weight of pulp, 10 ml of the hardwood preinoculum and 100 mlof 1× of the concentrate modified growth media.

Hardwood kraft pulp was also treated in second series by mixing 5 gramsdry weight of pulp, 10 ml of the hardwood preinoculum, 1.0 gram of xylanand 100 ml of the 1× concentrate modified growth media.

Softwood kraft pulp was treated in a third series by mixing 5 grams dryweight of pulp, 10 ml of softwood preinoculum and 100 ml of the 1×concentrate of the modified growth media.

Softwood kraft pulp was also treated in a fourth series by mixing 5grams dry weight of pulp, 10 ml of softwood preinoculum, 1.0 grams ofmannan and 100 ml of the 1× modified growth media.

Softwood kraft pulp was also treated in a fifth series similar to thefourth series except that 0.5 grams each of xylan and mannan wassubstituted for the 1.0 gram mannan.

A sixth, seventh, eighth, ninth and tenth series were prepared using 20ml of 2× concentrate modified growth media in place of the 100 ml of 1×growth media (resulting in a pulp consistency of 16.5% ).

Controls were run without the addition of xylan, mannan or combinationthereof at 4.5% and 16.5% softwood pulp and hardwood pulp.

Treatments were run for 15 and 50 days, after which Kappa Number wasevaluated. The results are given below in Table J (15 days) and Table K(50 days).

                  TABLE J                                                         ______________________________________                                        15 Days - Kappa Results                                                              Hardwood-Consistency                                                                        Softwood  Consistency                                    Series   4.5%      16.5%     4.5%    16.5%                                    ______________________________________                                        No extra 13.3      10.3      23.5    17.9                                     carbon                                                                        source                                                                        Plus Mannan                  21.5    17.4                                     Plus Xylan                                                                             13.9      14.5      --      --                                       Plus Mannan                                                                            --        --        22.1    16.4                                     and Xylan                                                                     Control  15        15        22      22                                       ______________________________________                                    

                  TABLE K                                                         ______________________________________                                        50 Days - Kappa Results                                                              Hardwood-Consistency                                                                        Softwood-Consistency                                     Series   4.5%      16.5%     4.5%    16.5%                                    ______________________________________                                        No extra 7         5.2       9.8     6.1                                      carbon                                                                        source                                                                        Plus Mannan                                                                            --        --        12      5.1                                      Plus Xylan                                                                             12        10.8      --      --                                       Plus Mannan                                                                            --        --        13.2    4.7                                      and Xylan                                                                     Control  15        15        22      22                                       ______________________________________                                    

The kappa number is the volume (in milliliters) of 0.1N potassiumpermanganate solution consumed by one gram of moisture-free pulp underthe conditions specified in this method. The results are corrected to50% consumption of the permanganate added. In determining Kappa numberin the foregoing examples, the following standard method which isincorporated herein by reference was used: TAPPI Test Methods, (Tappi,Atlanta, Ga.) Vol. 1, 1988 "Kappa number of pulp--T 236 cm 85".

What is claimed is:
 1. A culture composition comprising a biologicallypure culture of Scytinostroma galactinum strain F361 fungus which iscapable of producing manganese oxide in stationary liquid culture whenveratryl alcohol is present and when wood, is absent and a lignolyticenzyme inducer selected from the group consisting of an isolated andpurified lignin, a cellulosic substrate containing a bound lignincomponent and an aromatic ring-containing lignin component degradationproduct which is unbound to a cellulosic substrate.
 2. A culture ofclaim 1 in which the inducer is a processed or unprocessed wood in whichits lignin component has not been chemically treated.
 3. A culture ofclaim 1 in which the inducer is a lignin model compound.
 4. A culture ofclaim 1 in which the inducer is wood pulp in which the lignin componentis a chemically modified lignin component.
 5. A culture of claim 4 inwhich the inducer is unbleached kraft pulp.